LYMPHOTOXIN IS A LYMPHOKINE WHICH HAS POTENT ANTITUMOR ACTIVITY.

LYMPHOTOXIN IS A LYMPHOKINE WHICH HAS POTENT ANTITUMOR ACTIVITY. IT IS A GLYCOPROTEIN WITH A MOLECULAR WEIGHT OF 25,000 DALTONS. DUE TO THE MINUTE AMOUNT PRODUCED BY LYMPHOCYTES, EVALUATION OF ITS PHARMACEUTICAL POTENTIAL REQUIRES THAT IT BE CLONED AND EXPRESSED IN A SUITABLE HOST. IN ORDER TO OBTAIN THE CORRECT GLYCOSYLATION PATTERN, WE PROPOSE TO EXPRESS IT IN MAMMALIAN CELLS. IN PHASE I, WE PLAN TO CLONE THE FULL-LENGTH LYMPHOTOXIN CDNA. PERIPHERAL BLOOD LYMPHOCYTES WILL BE USED FOR POLY(A) RNA ISOLATION FROM WHICH CDNA WILL BE SYNTHESIZED. THE VECTOR WE CHOOSE TO USE FOR ESTABLISHING A CDNA LIBRARY IS THE SINGLE-STRANDED PHAGE FL. THE CDNA LIBRARY WILL BE SCREENED WITH CHEMICALLY SYNTHESIZED OLIGONUCLEOTIDES. THE FULL-LENGTH CDNA WILL BE ASSEMBLED WITH OVERLAPPING RECOMBINANT CLONES IF NECESSARY. IT WILL ALSO BE SEQUENCED. IN ADDITION, THE ACTIVITY ASSAY FOR LYMPHOTOXIN WILL BE SET UP IN PHASE I. FOR PHASE II, WE PROPOSE TO WORK ON EXPRESSING THE CDNA IN MAMMALIAN CELL CULTURE AND MAXIMIZING THE LEVEL OF EXPRESSION.